Epidemiology of Haemophilus influenzae in children on Lombok Island, Indonesia

The Haemophilus influenzae serotype b (Hib) conjugate vaccine routine immunization programme has been implemented for almost a decade; however, there is limited surveillance of H. influenzae carriage rates in the Indonesian population. H. influenzae was isolated from nasopharyngeal (NP) swab specimens of healthy children on Lombok Island, West Nusa Tenggara Province, Indonesia from 2018 to 2019. Serotyping was performed using quantitative polymerase chain reaction. We identified H. influenzae in 40 of the 96 (41.6 %) NP swab specimens. We identified 39 non-typeable H. influenzae (NTHi) isolates and 1 Hib isolate.


INTRODUCTION
Haemophilus influenzae is a Gram-negative coccobacillus that causes non-invasive diseases, such as otitis media and sinusitis, and invasive diseases, such as pneumonia and meningitis.Based on capsule polysaccharide expression, this pleomorphic bacterium is categorized as non-capsulated or non-typeable (non-typeable H. influenzae; NTHi) and capsulated or typeable, which includes six serotypes (a-f) [1,2].During the pre-vaccine era, serotype b (Hib) accounted for 95 % of all invasive diseases due to H. influenzae and was the most common cause of bacterial meningitis in children <5 years of age, with 83 % of cases occurring in children <2 years old.The burden of Hib disease remains significant, especially among children <5 years, even though the Hib vaccine has been introduced in routine infant immunization programmes in many countries [1,3].Additionally, an increasing number of non-Hib invasive diseases have been consistently reported in the post-Hib vaccine era [4].The Hib vaccine, as part of the pentavalent vaccine, was integrated into a routine immunization programme in Indonesia in 2013, but post-vaccination surveillance of H. influenzae cases is still very limited [5,6].
H. influenzae is primarily transmitted through direct contact with respiratory droplets from nasopharyngeal carriers [7].Nasopharyngeal H. influenzae carriers are considered the only reservoirs and transmission vehicles for invasive diseases.Reports showed that the rate of H. influenzae carriers is approximately 20 % in infants and >50 % in children aged 5-6 [8].This study investigated the prevalence of H. influenzae carriers in children in Lombok, several years after implementation of a routine Hib immunization programme.Additionally, the results of the current study likely reflect similar epidemiological trends in most regions in the country, which correlate with the public health and socioeconomic issues in Lombok Island.

METHODS
Archived nasopharyngeal (NP) swab specimens from a previous prospective cohort pneumococcal carriage study on Lombok Island, West Nusa Tenggara, Indonesia, conducted between March 2018 and June 2019, were used [9].The study enrolled healthy infants (2 months old) who were brought to the primary care facility for routine vaccination and were randomized into two groups: pneumococcal vaccine (PCV)-vaccinated group and the non-PCV-vaccinated group (control group).The two groups were matched based on age and home location.All recruited children were healthy when the NP swab specimens were obtained [9].In the current study, we randomly sampled 96 of 233 archived NP swab specimens from both the vaccine (64 specimens) and control (32 specimens) groups, which were obtained 6 months after the PCV vaccine booster was administered to the vaccine group.The sample size was calculated based on the sample size formula for estimating a single proportion with an approximate power of >0.95.
NP swab specimen-inoculated skim milk, tryptone, glucose and glycerol (STGG) medium was thawed, and 100 µl was transferred onto a supplemented chocolate agar plate (5 % sheep blood and vitox/isovitalex) with 20 U ml −1 bacitracin (CAPwB).Plates were incubated at 37 °C with 5 % CO 2 for 18-24 h.Suspect H. influenzae colonies were characterized as oxidase-positive; nonhaemolytic; opaque to grey colour; large, round, convex colonies; creamy texture with pungent indole smell [10].They appeared as Gram-negative coccobacilli with pleomorphic morphology.The X and V factor test was performed since H. influenzae only grows around the XV disc [10,11].A single colony-forming unit of confirmed H. influenzae isolate was restreaked, harvested and then stored in STGG medium at −80 °C as a stock isolate.All suspected H. influenzae colonies were confirmed by detecting the hpd gene and serotyped utilizing a real-time polymerase chain reaction.The gene targets for serotypes a, b, c, d, e and f are acsB, bcsB, ccsD, dcsE, ecsH and bexD, respectively, as previously described [11].
The laboratory results were summarized as the total prevalence of carriage and the distribution of serotypes among the positive results.We also reviewed relevant provenance data collected using a questionnaire developed in a previous study, which included the following variables: sex, nutritional status, number of people in the household and cigarette smoke exposure [9].
Based on polymerase chain reaction-based serotyping, 39 isolates were NTHi and only 1 was Hib.Among the positive specimens, 23 (57.5 %) and 17 (42.5 %) were from males and females, respectively (Table S1).We also found that 69 % of participants had been exposed to cigarette smoke in their households.
The prevalence of H. influenzae carriage in this study was higher than that reported in a previous pre-Hib vaccination era study (32 % of 484 subjects) in Lombok in 1997 (Table 1).However, the prevalence of Hib in our study (2.5 %, 1/40) was lower than that reported by this same Lombok 1997 study (14 %, 22/155) [12].Another study conducted in Bandung, Lombok and Padang in 2016 also found an H. influenzae carriage rate of 27.5 % in 302 subjects, which was lower than that in the current study, even though no Hib was detected, probably because of the implementation of a routine Hib immunization programme.A report on H. influenzae carriage rate among children with HIV infection in Jakarta found a significantly lower rate of 18 % [11].Recently, the prevalence of H. influenzae was reported to be 69.7 % (85/122) among schoolchildren with acute otitis media in Banyumas Regency, Central Java, Indonesia, with 3.5 and 1.2 % of the isolates being H. influenzae serotypes b and f, respectively [6] (Table 1).
The impact of the routine Hib immunization programme, which has been implemented for almost a decade, is reflected in the reduced rate of Hib carriage.However, the overall increased prevalence of H. influenzae carriage found in the current study, with predominantly NTHi, remains a concern.NTHi is the major upper respiratory commensal that occasionally causes both noninvasive diseases, such as acute otitis media, sinusitis and conjunctivitis, and invasive diseases (e.g.meningitis and septicaemia) [13].A convincing body of evidence has shown a shift in invasive cases caused by non-type b H. influenzae, especially NTHi, in many countries [4,5].Therefore, NTHi, as a common group in carriers, can be considered to be a pool of potentially pathogenic strains that can lead to invasive diseases in susceptible individuals.Unfortunately, in many developing countries such as Indonesia, it is difficult to detect the H. influenzae serotype that causes invasive diseases, or even to detect the bacterium at all, as there are only a few laboratories capable of isolating and identifying H. influenzae.Response:Thank you for your input.The detection of hpdgene was conducted using real time PCR (RT-PCR) therefore gel electrophoresis step was not needed.However, we have CT value data and we have provided this as a supplementary table S1for you to review.
12. Question:Set the font size of references as per journal format Response:Thank you for your review.We have set the font size of references per journal format.

Reviewer 2
This is a brief report of an H. influenzae carriage study on Lombok Island, Indonesia.The broader public health relevance of the study needs to be explained more clearly, and the estimate of the carriage prevalence should be given a 95% confidence interval.I have a number of other editing suggestions below.
1. Question:(Introduction) It would be helpful to give an explanation of why the prevalence of H. influenzae carriage on Lombok Island is of public health interest in the rest of Indonesia or beyond.Clearly, it is important on Lombok Island itself.But what do the findings mean for people who are not on Lombok Island?
Response:Thank you for your input.We have added a further explanation to this in the introduction section, specifically in line 84-85 and 90-94.In principal, there are three reasons why the current H. influenzae carriage in Lombok is of public health interest: • The first and only study on H. influenzae related disease was conducted in Lombok (Gessner et al., 1998), followed by an evaluation of the carriage rate (Gessner et al., 2005) in children under 2 years old.It is logical to re-evaluate this rate 20 years later, especially after the nation-wide Hib immunisation program (Hib vaccine coverage in Lombok 2017 was 87 -100% for the first three doses, and 56 -90% for the 4th dose) (NTB Satu Data: Hib immunization coverage, 2017).
• While Dunne et al. also evaluated this in 2016, identification of H. influenzae carriage was solely based on comersialised molecular diagnostic kit, of which the target gene was not mentioned.
• Lombok Island demographic and epidemiology reflects most regions in Indonesia, which are usually a mixture of both urban and rural areas.Therefore, major vaccination impact studies and implementation started in Lombok Island (e.g.Hib and more recently, PCV13).
2. Question:(line 54) Instead of "which comprising", it might be better to say "which has" or "which contains".
Response:Thank you for your correction.We intended to describe that there are six different serotypes for capsulated H. influenzae group, so we decided to change "which comprising" to "which includes".
3. Question:(lines 77-79) From the description, it appears that the nasal swabs were taken from symptomatic children.If so, the estimated prevalence from this study would be much higher than a prevalence estimate based on a representative sample of children.
Response:Thank you for your comment.We realised that the description given in this line was unclear and irrelevant.Below is our explanation: We did four swabs for each children.All swabs were taken when the children visited primary care facility to receive basic immunisation.Basic immunisation refers to the vaccines included in standard, national programme (e.g.HB-Hib-DPT, OPV, Measles), so despite their group in the study (i.e.receive PCV or not), they still received their basic immunisation.All basic vaccines were only given if the children did not show any symptoms and this was the standard/routine practice.If at any point during the follow-up they were symptomatic, they would be asked to return after they recovered for a catch-up immunisation and the "catch-up" swabs.These catch-up immunisation was for them to receive any of the basic vaccines the children should have gotten but couldn't because they were sick.Therefore, at any point, all children were only swabbed if they did not have any health complaints (asymptomatic).
Please kindly note that only among the last (4th) swabs was selected for the H. influenzae carriage study.
To simplify this in the manuscript,we have revised to previous description into "All recruited children were healthy when the nasopharyngeal swab specimen was obtained".(line 99) 4. Question:(line 79) The catch-up vaccination for children who became unwell needs more explanation.Was it a vaccination given to children in the control group who became unwell or did children in the vaccination group also receive an additional vaccination if they became sick?
Response:Thank you for your question.As we mentioned in our response to question 3 above, our previous description was unclear.Please kindly refer to our response above.Response:Thank you for your feedback.We have added more details about the specimen selection and sampling method in line 99-104.
6. Question:(lines 91-93) The plural should be used: "All suspected H. influenzae colonies were confirmed . . ." Response:Thank you for your feedback, this now has been corrected.7. Question:(lines 96-97) The first sentence of the Results & Discussion is not a complete sentence.It should say something like: "Ninety-six swab samples were collected . . .".In the second sentence, it might be better to say "isolated" instead of "identified".
Response:Thank you for your feedback, this now has been corrected.8. Question:(lines 98-100) It might be better to say something like: "Based on polymerase chain reaction-based serotyping, 39 isolates were NTHi and only one was type b." Response:Thank you for your feedback, this now has been corrected.9. Question:(line 100) The percentages male and female among positive cases should probably be calculated with the number of positive cases (40) as the denominator, not the entire sample (98).The current percentages show the proportion of the entire sample that are male positive cases and female positive cases, which does not seem to match the description in the text.
Response:Thank you for your feedback, this now has been corrected.10.Question:(lines 102-104) The comparison of the current study to the 1997 study seems to be comparing apples and oranges.The 41.6% carriage prevalence in the current study includes both type-b and NTHi, but the 1997 study seems to have found.
Response:The 1997 study showed the carriage prevalence was 32% (155 isolates from 484 children.The encapsulated H. influenzae was 22% (34/155) with Hib was 14% (22/155), type e 3% (4/155), type d and f 2% (3/155), 0.6% (1/155) for type a and c.We miscalculated the Hib prevalence of the 1997 study.We have corrected the sentence.11.Question:(Table 1) The n for each prevalence estimate is given in the fourth column, so it is not necessary to repeat it in the fifth column (e.g., "40/96").These prevalence estimates should be given 95% confidence intervals wherever possible.In particular, the prevalence estimate from the current study needs to have a confidence interval.
Response:Thank you for your suggestion.We have deleted the denominator in the fifth column.We have added the confidence intervals when it is available.12. Question:(Table 1) It would help to arrange the rows in the table in decreasing chronological order (most recent at the top and oldest at the bottom).
Response:Thank you for your input.We have put the table in chronological order (the most recent at the top followed by the oldest at the bottom).
13. Question:(Discussion) The argument about the potential danger of invasive disease from NTHi is important, but it seems to be missing an important nuance.If vaccination has successfully controlled type-b H. influenzae, then it is not unexpected that some of this will be replaced by NTHi.The important public health question is whether the rate of invasive disease has gone up or down.The results of the present study do not answer this question.
Response:We could not estimate whether the rate of invasive disease is gone up or down since the H. influenzae invasive disease rate is extremely limited in Indonesia.However, we presented carriage rate thay might be the potential of the invasive disease.Our serotype distribution results predict the serotypes that might be arise causing invasive disease in the future.The high carriage of NTHi could be the risk for the Hi invasive disease rising since they are not included in the Hib routine immunization program.This explanation has been added in the last paragraph (line 143-159).

Date report received: 24 May 2023 Recommendation: Major Revision
Comments: This is a brief report of an H. influenzae carriage study on Lombok Island, Indonesia.The broader public health relevance of the study needs to be explained more clearly, and the estimate of the carriage prevalence should be given a 95% confidence interval.I have a number of other editing suggestions below.Comments 1. (Introduction) It would be helpful to give an explanation of why the prevalence of H. influenzae carriage on Lombok Island is of public health inter-est in the rest of Indonesia or beyond.Clearly, it is important on Lombok Island itself.But what do the findings mean for people who are not on Lombok Island? 2. (line 54) Instead of "which comprising", it might be better to say "which has" or "which contains".3. (lines 77-79) From the description, it appears that the nasal swabs were taken from symptomatic children.If so, the estimated prevalence from this study would be much higher than a prevalence estimate based on a representative sample of children.4. (line 79) The catch-up vaccination for children who became unwell needs more explanation.Was it a vaccination given to children in the control group who became unwell or did children in the vaccination group also receive an additional vaccination if they became sick? 5. (lines 79-82) The number of children in the original study should be specified.It is not clear what proportion of the children in the original cohort study is represented by the 96 children in the current study.The selection process for the 96 samples also needs to be explained because it affects the interpretation of the prevalence estimate.6. (lines 91-93) The plural should be used: "All suspected H. influenzae colonies were confirmed . . ." 7. (lines 96-97) The first sentence of the Results & Discussion is not a complete sentence.It should say something like: "Ninety-six swab samples were collected . . .".In the second sentence, it might be better to say "isolated" instead of "identified".8. (lines 98-100) It might be better to say something like: "Based on polymerase chain reaction-based serotyping, 39 isolates were NTHi and only one was type b." 9. (line 100) The percentages male and female among positive cases should probably be calculated with the number of positive cases (40) as the denominator, not the entire sample (98).The current percentages show the proportion of the entire sample that are male positive cases and female positive cases, which does not seem to match the description in the text.10. (lines 102-104) The comparison of the current study to the 1997 study seems to be comparing apples and oranges.The 41.6% carriage prevalence in the current study includes both type-b and NTHi, but the 1997 study seems to have found.11. (Table 1) The n for each prevalence estimate is given in the fourth column, so it is not necessary to repeat it in the fifth column (e.g., "40/96").These prevalence estimates should be given 95% confidence intervals wherever possible.In particular, the prevalence estimate from the current study needs to have a confidence interval.12. (Table 1) It would help to arrange the rows in the table in decreasing chronological order (most recent at the top and oldest at the bottom).13. (Discussion) The argument about the potential danger of invasive disease from NTHi is important, but it seems to be missing an important nuance.If vaccination has successfully controlled type-b H. influenzae, then it is not unexpected that some of this will be replaced by NTHi.The important public health question is whether the rate of invasive disease has gone up or down.The results of the present study do not answer this question.

Please rate the quality of the presentation and structure of the manuscript Satisfactory
To what extent are the conclusions supported by the data?Partially support

5 .
Question:(lines 79-82) The number of children in the original study should be specified.It is not clear what proportion of the children in the original cohort study is represented by the 96 children in the current study.The selection process for the 96 samples also needs to be explained because it affects the interpretation of the prevalence estimate.

VERSION 1 Editor
recommendation and comments https://doi.org/10.1099/acmi.0.000609.v1.5 © 2023 Tolman L. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Lindsey Tolman; University at Albany, UNITED STATES Date report received: 25 May 2023 Recommendation: Minor Amendment Comments: This study would be a valuable contribution to the existing literature.The reviewers have highlighted minor concerns with the work presented.Please ensure that you address their comments.Reviewer 2 recommendation and comments https://doi.org/10.1099/acmi.0.000609.v1.4 © 2023 Anonymous.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.

Table 1 .
Prevalence of H. influenzae carriage among Indonesian children and the adult population *In this study, only 34 out of 155 H. influenzae isolates were serotyped.

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World Health Organization.Prevention (U.S.) C for DC and.Laboratory methods for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae: WHO manual.World Health Organization; 2011 11.Safari D, Lestari AN, Khoeri MM, Tafroji W, Giri-Rachman EA, et al.Nasopharyngeal carriage and antimicrobial susceptibility profile of Haemophilus influenzae among patients infected with HIV in Jakarta, Indonesia.Access Microbiol 2020;2:acmi000165.12. Gessner BD, Sutanto A, Steinhoff M, Soewignjo S, Widjaya A, et al.A population-based survey of Haemophilus influenzae type b nasopharyngeal carriage prevalence in Lombok Island, Indonesia.Author feedback says our Editors are 'thorough and fair' and 'patient and caring'. 5. Increase your reach and impact and share your research more widely.
The evidence of increasing H. influenzae carriage rates should provoke consideration for routine active surveillance of H. influenzae diseases and improve laboratories' capacity to identify and characterize them based on serotypes.Additionally, based on the serotype distribution results of our study, it can be predicted that NTHi will be the dominant group underlying invasive H. influenzae infection 1.When you submit to our journals, you are supporting Society activities for your community.2. Experience a fair, transparent process and critical, constructive review.3.If you are at a Publish and Read institution, you'll enjoy the benefits of Open Access across our journal portfolio.4.